EP3042953A1 - Procédé d'utilisation d'hexénol glycosyl transférase - Google Patents

Procédé d'utilisation d'hexénol glycosyl transférase Download PDF

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EP3042953A1
EP3042953A1 EP14831646.6A EP14831646A EP3042953A1 EP 3042953 A1 EP3042953 A1 EP 3042953A1 EP 14831646 A EP14831646 A EP 14831646A EP 3042953 A1 EP3042953 A1 EP 3042953A1
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Prior art keywords
amino acid
protein
acid sequence
hexenol
seq
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German (de)
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EP3042953A4 (fr
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Eiichiro Ono
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Suntory Holdings Ltd
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Suntory Holdings Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)

Definitions

  • the present invention relates to a method for producing a hexenol glycoside, a transformant that highly expresses hexenol glycosyltransferase, as well as a hexenol glycoside produced by the above method and use thereof.
  • the present invention also relates to a plant modified to suppress the expression of a protein having glycosylation activity on a hexenol compound and use thereof.
  • Green leaf volatiles typified by cis -3-hexenol are used to collectively refer to plant aroma components containing 6 carbon atoms, which constitute an aroma that is released from plants upon feeding damage and/or mechanical injury (Non-patent Document 1). Green leaf volatiles have repellent or attractive activity on insects or larvae thereof and are considered to be responsible for physiological roles as allelochemicals (Non-patent Document 2).
  • Green leaf volatiles are known to be generated by being liberated from linolenic acid in chloroplasts, although they are reported to be partially pooled within plant cells in the form of glycosides such as glucoside and primeveroside (Non-patent Document 3). These disaccharide glycosides including primeveroside are also known to be generated through a pathway where they are specifically hydrolyzed by the action of primeverosidase to release their aglycons, i.e., green leaf volatiles (Non-patent Document 4). However, there is no knowledge about enzymes and molecular mechanisms for glycosylation of green leaf volatiles and their accumulation within plant cells.
  • the inventors of the present invention have succeeded in identifying enzymes catalyzing the glycosylation reaction of hexenol in Camellia sinensis, hop ( Humulus lupulus ), stevia ( Stevia rebaudiana ), grape ( Vitaceae vitis ), sweet potato ( Ipomoea batatas ), Arabidopsis thaliana and snapdragon ( Antirrhinum majus ), as well as gene sequences encoding these enzymes.
  • the present invention is based on the above finding.
  • the present invention is as follows.
  • the method or composition of the present invention enables the production of hexenol glycosides with high efficiency.
  • the inventors of the present invention have elucidated, ahead of others, that specific proteins have glycosylation activity on hexenol.
  • polynucleotides and enzymes can be obtained by procedures as described later in the Example section, known genetic engineering procedures, known synthesis procedures, etc.
  • the present invention provides a method for producing a hexenol glycoside, which comprises the step of reacting a protein having glycosylation activity on hexenol (hereinafter referred to as "the protein of the present invention") with a UDP-sugar and a hexenol molecule to cause glycosylation of the hexenol molecule.
  • the present invention also provides the use of the protein of the present invention in the production of hexenol glycosides.
  • the present invention also provides the protein of the present invention for use in the production of hexenol glycosides.
  • the protein of the present invention is more specifically any one selected from the group consisting of (a) to (c) shown below:
  • amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 is intended in some embodiment to mean "the amino acid sequence shown in SEQ ID NO: 2 or 4," “the amino acid sequence shown in SEQ ID NO: 6, 8, 10 or 12,” “the amino acid sequence shown in SEQ ID NO: 14, 16 or 18,” “the amino acid sequence shown in SEQ ID NO: 20, 22 or 24,” “the amino acid sequence shown in SEQ ID NO: 26,” "the amino acid sequence shown in SEQ ID NO: 28 or 30” or "the amino acid sequence shown in SEQ ID NO: 32.”
  • the above protein (b) or (c) is typically a mutant of the naturally occurring polypeptide shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32, although other examples include those which may be artificially obtained by site-directed mutagenesis as described in " Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor Laboratory Press 2001 ,” “ Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997 ,” “ Nuc. Acids. Res., 10, 6487 (1982 ),” “ Proc. Natl. Acad. Sci. USA, 79, 6409 (1982 ),” “ Gene, 34, 315 (1985 ),” " Nuc. Acids. Res., 13, 4431 (1985 ),” “ Proc. Natl. Acad. Sci. USA, 82, 488 (1985 ),” etc.
  • protein which consists of an amino acid sequence with deletion, substitution, insertion and/or addition of 1 to 40 amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 and which has glycosylation activity on hexenol is intended to include proteins which consist of an amino acid sequence with deletion, substitution, insertion and/or addition of, e.g., 1 to 40 amino acid residues, 1 to 39 amino acid residues, 1 to 38 amino acid residues, 1 to 37 amino acid residues, 1 to 36 amino acid residues, 1 to 35 amino acid residues, 1 to 34 amino acid residues, 1 to 33 amino acid residues, 1 to 32 amino acid residues, 1 to 31 amino acid residues, 1 to 30 amino acid residues, 1 to 29 amino acid residues, 1 to 28 amino acid residues, 1 to 27 amino acid residues, 1 to 26 amino acid residues, 1 to 25 amino acid residues, 1 to 24 amino acid residues, 1 to 23 amino acid residues, 1
  • examples of such proteins include those which have an amino acid sequence sharing a sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more with the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 and which have glycosylation activity on hexenol. In general, a larger value is more preferred for the above sequence identity.
  • glycosylation activity on hexenol is intended to mean the ability to add a sugar included in a UDP-sugar to a hydroxy group (-OH group) in hexenol serving as an aglycon (i.e., glycosylation).
  • aglycon i.e., glycosylation
  • Glycosylation activity on hexenol can be confirmed by reacting the protein of the present invention with a UDP-sugar and a hexenol molecule to detect a hexenol glycoside.
  • the reaction of the protein of the present invention with a UDP-sugar and a hexenol molecule may be conducted by incubation at a temperature of 20°C to 40°C in a system (preferably a neutral buffer of pH 6.0 to 8.0 (e.g., sodium phosphate buffer or potassium phosphate buffer)) which contains the protein of the present invention in an amount of 1 to 500 ng (preferably 50 to 200 ng, most preferably 100 ng), a UDP-sugar (e.g., UDP-glucose) at 1 to 1000 ⁇ M (preferably 100 to 700 ⁇ M, most preferably 500 ⁇ M) and hexenol (e.g., cis -3-hexenol) at 1 to 500 ⁇ M (preferably 100 to 500 ⁇ M, most preferably 250 ⁇ M).
  • Glycosylation reaction is normally completed within about 1 minute to about 12 hours.
  • the above hexenol may be purified and analyzed by known procedures such as LC-MS analysis (liquid chromatography-mass spectrometry), etc. Namely, if the purified hexenol shows a glycoside peak, it can be determined that hexenol was glycosylated by the activity of the protein of the present invention.
  • the protein of the present invention particularly those shown below have glycosylation activity not only on hexenol, but also on geraniol, eugenol, benzyl alcohol, 2-phenylethanol and linalool ( Figure 7 ):
  • proteins (w) and (x) are equivalent to proteins (b') and (c') shown below, respectively:
  • the present invention also provides a method for glycosylation of any one or more substrates selected from the group consisting of hexenol, geraniol, eugenol, benzyl alcohol, 2-phenylethanol and linalool.
  • the expression "glycosylation activity on any one or more substrates selected from the group consisting of hexenol, geraniol, eugenol, benzyl alcohol, 2-phenylethanol and linalool” is intended to mean the ability to add a sugar included in a UDP-sugar to a hydroxy group (-OH group) in any one or more substrates selected from the group consisting of geraniol, eugenol, benzyl alcohol, 2-phenylethanol and linalool (hereinafter referred to as "the substrate of the present invention") being in an aglycon state (i.e., glycosylation).
  • the substrate of the present invention There is no particular limitation on the position of the hydroxy group where sugar addition occurs.
  • Glycosylation activity of the present invention on its substrate can be confirmed by reacting the protein of the present invention with a UDP-sugar and the substrate molecule of the present invention and detecting a hexenol glycoside.
  • the reaction of the protein of the present invention with a UDP-sugar and the substrate molecule of the present invention may be conducted by incubation at a temperature of 20°C to 40°C in a system (preferably a neutral buffer of pH 6.0 to 8.0 (e.g., sodium phosphate buffer or potassium phosphate buffer)) which contains the protein of the present invention in an amount of 1 to 500 ng (preferably 50 to 200 ng, most preferably 100 ng), a UDP-sugar (e.g., UDP-glucose) at 1 to 1000 ⁇ M (preferably 100 to 700 ⁇ M, most preferably 500 ⁇ M) and the substrate of the present invention at 1 to 500 ⁇ M (preferably 100 to 500 ⁇ M, most preferably 250 ⁇ M).
  • Glycosylation reaction is normally completed within about 1 minute to about 12 hours.
  • the above substrate may be purified and analyzed by known procedures such as LC-MS analysis (liquid chromatography-mass spectrometry), etc. Namely, if the purified substrate of the present invention shows a glycoside peak, it can be determined that the substrate of the present invention was glycosylated by the activity of the protein of the present invention.
  • LC-MS analysis liquid chromatography-mass spectrometry
  • Deletion, substitution, insertion and/or addition of one or several amino acid residues in the amino acid sequence of the protein of the present invention is intended to mean that deletion, substitution, insertion and/or addition of one or several amino acid residues occurs at any one or more positions in the same sequence, and two or more of deletion, substitution, insertion and addition may occur at the same time.
  • Group A leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine;
  • Group B aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid;
  • Group C asparagine, glutamine;
  • Group D lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid;
  • Group E proline, 3-hydroxyproline, 4-hydroxyproline;
  • Group F serine, threonine, homoserine;
  • Group G phenylalanine
  • the protein of the present invention may be obtained by being expressed from a polynucleotide encoding it (see “the polynucleotide of the present invention” described later) in appropriate host cells, it may also be prepared by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method). Alternatively, the protein of the present invention may also be chemically synthesized with peptide synthesizers commercially available from Advanced Automation Peptide Protein Technologies, Perkin Elmer, Protein Technologies, PerSeptive, Applied Biosystems, SHIMADZU, etc.
  • hexenol refers to hexene substituted with one or more -OH groups (molecular formula: C 6 H 12 O).
  • the hexene skeleton may be in any form, i.e., linear, branched or cyclic, preferably linear.
  • hexenol may have a carbon-carbon double bond at any position, although it preferably has a double bond between the carbon at the 3-position and the carbon at the 4-position.
  • the hexene skeleton may be in either cis- or trans-configuration.
  • the number of -OH groups is preferably 1.
  • the -OH group is preferably attached to the terminal carbon atom of the linear or branched hexene skeleton, more preferably attached to the terminal carbon atom of the linear hexene skeleton.
  • hexenol include, but are not limited to, cis -3-hexenol ( ci s-3-hexen-1-ol) and trans -2-hexenol ( trans -2-hexen-1-ol).
  • hexenol is used interchangeably with “leaf alcohol” in some cases.
  • leaf alcohol include cis -3-hexenol and trans -2-hexenol ( trans -2-hexen-1-ol).
  • benzyl alcohol is also referred to as phenylmethanol.
  • 2-phenylethanol refers to a compound which is also referred to as phenethyl alcohol, benzyl carbinol, ⁇ -hydroxyethylbenzene, ⁇ -phenylethyl alcohol, phenethanol, phenylethyl alcohol, ⁇ -phenethyl alcohol, ⁇ -phenylethanol, ⁇ -phenethanol, phenylethanol, 2-phenethanol or ⁇ -phenethylol.
  • UDP-sugar refers to a uridine diphosphate (UDP)-conjugated sugar, preferably a UDP-conjugated hexose (UDP-hexose). Examples include, but are not limited to, UDP-glucose, UDP-mannose and UDP-galactose. A preferred UDP-sugar is UDP-glucose.
  • hexenol glycoside is intended to mean hexenol having a sugar (derived from a UDP-sugar) added to at least one or more -OH groups.
  • cis -3-hexenol when cis -3-hexenol is reacted with UDP-glucose in the presence of the protein of the present invention, cis -3-hexenyl monoglucoside is generated as a hexenol glycoside (see the reaction scheme shown below).
  • the method of the present invention for producing a hexenol glycoside comprises the step of reacting the protein of the present invention with a UDP-sugar and hexenol to cause glycosylation of the hexenol.
  • the method of the present invention may further comprise the step of purifying the hexenol glycoside generated in the above step.
  • the hexenol glycoside can be purified by known techniques such as extraction with an appropriate solvent (an aqueous solvent such as water or an organic solvent such as alcohol, ether or acetone), a gradient between an organic solvent (e.g., ethyl acetate) and water, high performance liquid chromatography (HPLC), gas chromatography, time-of-flight mass spectrometry (TOF-MS), ultra (high) performance liquid chromatography (UPLC), etc.
  • an appropriate solvent an aqueous solvent such as water or an organic solvent such as alcohol, ether or acetone
  • HPLC high performance liquid chromatography
  • TOF-MS time-of-flight mass spectrometry
  • UPLC ultra (high) performance liquid chromatography
  • a glycoside of the substrate of the present invention is intended to mean the substrate of the present invention having a sugar (derived from a UDP-sugar) added to at least one or more -OH groups.
  • a method for producing a glycoside of the substrate of the present invention comprises the step of reacting the protein of the present invention with a UDP-sugar and the substrate of the present invention to cause glycosylation of the hexenol (substrate?).
  • the method of the present invention may further comprise the step of purifying the glycoside of the substrate of the present invention generated in the above step.
  • the glycoside of the substrate of the present invention can be purified by known techniques such as extraction with an appropriate solvent (an aqueous solvent such as water or an organic solvent such as alcohol, ether or acetone), a gradient between an organic solvent (e.g., ethyl acetate) and water, high performance liquid chromatography (HPLC), gas chromatography, time-of-flight mass spectrometry (TOF-MS), ultra (high) performance liquid chromatography (UPLC), etc.
  • an appropriate solvent an aqueous solvent such as water or an organic solvent such as alcohol, ether or acetone
  • HPLC high performance liquid chromatography
  • TOF-MS time-of-flight mass spectrometry
  • UPLC ultra (high) performance liquid chromatography
  • Hexenol glycosides may also be produced using the protein of the present invention within cells such as those of bacteria (e.g., E. coli or yeast), plants, insects, non-human mammals, etc.
  • a polynucleotide encoding the protein of the present invention (see “the polynucleotide of the present invention” described later) may be introduced into host cells derived from bacteria, plants, insects, non-human mammals or the like to cause expression of the protein of the present invention, followed by reacting the protein of the present invention with UDP-sugars and hexenol present within the above cells to produce hexenol glycosides.
  • the protein of the present invention is a recombinant protein generated by being expressed from a polynucleotide sequence encoding the protein in host cells.
  • a non-human transformant obtained by introduction of a gene encoding the protein of the present invention is expected to be rich in hexenol glycosides when compared to the wild-type counterpart.
  • the present invention therefore provides a non-human transformant transformed with a polynucleotide of any one selected from the group consisting of (a) to (e) shown below (hereinafter referred to as “the polynucleotide of the present invention") (such a transformant is hereinafter referred to as “the transformant of the present invention”):
  • nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31 is intended in some embodiment to mean "the nucleotide sequence shown in SEQ ID NO: 1 or 3," “the nucleotide sequence shown in SEQ ID NO: 5, 7, 9 or 11,” “the nucleotide sequence shown in SEQ ID NO: 13, 15 or 17,” “the nucleotide sequence shown in SEQ ID NO: 19, 21 or 23,” “the nucleotide sequence shown in SEQ ID NO: 25,” “the nucleotide sequence shown in SEQ ID NO: 27 or 29” or “the nucleotide sequence shown in SEQ ID NO: 31.”
  • amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 is intended in some embodiment to mean "the amino acid sequence shown in SEQ ID NO: 2 or 4," “the amino acid sequence shown in SEQ ID NO: 6, 8, 10 or 12,” “the amino acid sequence shown in SEQ ID NO: 14, 16 or 18,” “the amino acid sequence shown in SEQ ID NO: 20, 22 or 24,” “the amino acid sequence shown in SEQ ID NO: 26,” “the amino acid sequence shown in SEQ ID NO: 28 or 30” or “the amino acid sequence shown in SEQ ID NO: 32.”
  • polynucleotide of the present invention also includes polynucleotides shown below:
  • polynucleotide is intended to mean DNA or RNA.
  • polynucleotide which is hybridizable under high stringent conditions is intended to mean, for example, a polynucleotide that can be obtained by means of colony hybridization, plaque hybridization, Southern hybridization or other hybridization techniques using, as a probe, the whole or a part of a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31 or of a polynucleotide consisting of a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32.
  • high stringent conditions refers to, for example, but is not limited to, conditions of (1) 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 50°C, (2) 0.2 ⁇ SSC, 0.1% SDS, 60°C, (3) 0.2 ⁇ SSC, 0.1% SDS, 62°C, (4) 0.2 ⁇ SSC, 0.1% SDS, 65°C, or (5) 0.1 ⁇ SSC, 0.1% SDS, 65°C. Under these conditions, it can be expected that DNA having a higher sequence identity is efficiently obtained at a higher temperature.
  • the stringency of hybridization would be affected by a plurality of factors, including temperature, probe concentration, probe length, ionic strength, reaction time, salt concentration and so on. Those skilled in the art would be able to achieve the same stringency by selecting these factors as appropriate.
  • hybridization may be accomplished in accordance with the protocol attached to the kit, i.e., a membrane may be incubated overnight with a labeled probe and then washed with a primary washing buffer containing 0.1% (w/v) SDS under conditions of 55°C to 60°C to detect the hybridized DNA.
  • a commercially available reagent e.g., PCR labeling mix (Roche Diagnostics)
  • DIG digoxigenin
  • a probe during probe preparation based on the whole or a part of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31 or of a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32
  • a DIG nucleic acid detection kit may be used for detection of hybridization.
  • hybridizable polynucleotides include DNAs sharing a sequence identity of 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 % or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more with DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31 or with DNA encoding the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32, as
  • sequence identity of amino acid sequences or nucleotide sequences can be determined by using FASTA ( Science 227 (4693): 1435-1441, (1985 )) or the algorithm of Karlin and Altschul, BLAST (Basic Local Alignment Search Tool) ( Proc. Natl. Acad. Sci. USA 872264-2268, 1990 ; Proc Natl Acad Sci USA 90: 5873, 1993 ). Based on the algorithm of BLAST, programs called blastn, blastx, blastp, tblastn and tblastx have been developed ( Altschul SF, et al.: J Mol Biol 215: 403, 1990 ).
  • blastn is used for nucleotide sequence analysis
  • blastp is used for amino acid sequence analysis
  • BLAST and Gapped BLAST programs are used, default parameters in each program may be used.
  • the above polynucleotides according to the present invention can be obtained by known genetic engineering procedures or known synthesis procedures.
  • the polynucleotide of the present invention is preferably introduced into a host in a state of being inserted into an appropriate expression vector.
  • An appropriate expression vector is generally configured to comprise:
  • Such an expression vector may be prepared in any manner, for example, by techniques using plasmids, phages or cosmids, etc.
  • a promoter sequence may be selected as appropriate for the type of host cells in order to ensure expression of the polynucleotide of the present invention, and this promoter and the polynucleotide of the present invention may then be integrated into various plasmids or the like for use as expression vectors.
  • the expression vector of the present invention contains an expression control region(s) (e.g., a promoter, a terminator and/or a replication origin), depending on the type of host into which the expression vector is to be introduced.
  • Promoters for use in bacterial expression vectors may be commonly used promoters (e.g., trc promoter, tac promoter, lac promoter).
  • promoters for use in yeast include, for example, glyceraldehyde triphosphate dehydrogenase promoter, PH05 promoter and so on, while promoters for use in filamentous fungi include, for example, amylase, trpC and so on.
  • promoters used to express a desired gene in plant cells include cauliflower mosaic virus 35S RNA promoter, rd29A gene promoter, rbcS promoter, and mac-1 promoter that is configured to have the enhancer sequence of the above cauliflower mosaic virus 35S RNA promoter at the 5'-side of Agrobacterium -derived mannopine synthase promoter sequence.
  • the expression vector preferably comprises at least one selection marker.
  • drug resistance markers hygromycine, zeocin
  • geneticin resistance gene G418r
  • herbicide resistance gene Sur B
  • copper resistance gene CUP1
  • cerulenin resistance genes fas2m, PDR4
  • an expression vector comprising the polynucleotide of the present invention may be introduced into a host to transform the host, by way of example.
  • Host cells used for this purpose may be of any type, and conventionally known various types of cells can be used preferably. Specific examples include bacteria such as E . coli, yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe ) and plant cells, etc.
  • culture media and conditions appropriate for the above host cells are well known in the art.
  • the organism to be transformed may be of any type, and examples include various types of microorganisms or plants as listed above for host cells.
  • transformation may be accomplished by, but is not limited to, electroporation ( Mackenxie, D. A. et al., Appl. Environ. Microbiol., vol. 66, p. 4655-4661, 2000 ), particle delivery method (described in JP 2005-287403 A entitled “Breeding Method of Lipid Producing Fungi"), spheroplast method ( Proc. Natl. Acad. Sci. USA, vol. 75, p. 1929, 1978 ), lithium acetate method ( J. Bacteriology, vol. 153, p. 163, 1983 ), and other methods as described in Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual.
  • the transformant may be a plant transformant.
  • the plant transformant according to this embodiment may be obtained by introducing a recombinant vector comprising the polynucleotide of the present invention into a plant such that a polypeptide encoded by this polynucleotide can be expressed.
  • any recombinant expression vector may be used for transformation of a whole plant as long as it is a vector allowing the polynucleotide of the present invention to be expressed within the plant.
  • a vector include those having a promoter which drives constitutive expression of a desired polynucleotide within plant cells or those having a promoter whose activation is induced by external stimulation.
  • Examples of a promoter which drives constitutive expression of a desired polynucleotide within plant cells include cauliflower mosaic virus 35S RNA promoter, rd29A gene promoter, rbcS promoter, mac-1 promoter, etc.
  • Examples of a promoter whose activation is induced by external stimulation include mouse mammary tumor virus (MMTV) promoter, tetracycline-responsive promoter, metallothionein promoter and heat shock protein promoter, etc.
  • MMTV mouse mammary tumor virus
  • tetracycline-responsive promoter tetracycline-responsive promoter
  • metallothionein promoter metallothionein promoter
  • heat shock protein promoter etc.
  • to drive inducible expression of a desired polynucleotide within plant cells upon external stimulation it is possible to use a stress-inducible promoter, a high temperature- or low temperature-inducible promoter, etc.
  • a promoter for a gene specifically expressed in the intended organ it is possible to use a promoter for a gene specifically expressed in the intended organ.
  • the plant to be transformed in the present invention is intended to mean any of a whole plant, a plant organ (e.g., leaf, petal, stem, root, seed), a plant tissue (e.g., epidermis, phloem, parenchyma, xylem, vascular bundle, palisade tissue, spongy parenchyma) or a plant cultured cell, or alternatively, various forms of plant cells (e.g., suspension cultured cells), a protoplast, a leaf section, a callus and so on.
  • the plant used for transformation may be of any type, belonging to either monocotyledons or dicotyledons.
  • transformation techniques known to those skilled in the art may be used (e.g., Agrobacterium-mediated method, gene gun method, PEG-mediated method, electroporation).
  • Agrobacterium-mediated method and direct gene transfer into plant cells are well known.
  • the constructed plant expression vector may be introduced into an appropriate Agrobacterium strain (e.g., Agrobacterium tumefaciens ) and this strain may then be infected into a leaf section cultured under sterile conditions, e.g., in accordance with the leaf disk method ( Hirofumi Miyauchi, Manuals for Plant Genetic Engineering (1990) pages 27-31, Kodansha Scientific Ltd., Tokyo ) to thereby obtain a transgenic plant.
  • an appropriate Agrobacterium strain e.g., Agrobacterium tumefaciens
  • this strain may then be infected into a leaf section cultured under sterile conditions, e.g., in accordance with the leaf disk method ( Hirofumi Miyauchi, Manuals for Plant Genetic Engineering (1990) pages 27-31, Kodansha Scientific Ltd., Tokyo ) to thereby obtain a transgenic plant.
  • Hirofumi Miyauchi Manuals for Plant Genetic Engineering (1990) pages 27-31, Ko
  • an expression vector is first introduced into Agrobacterium, and the transformed Agrobacterium is then introduced into plant cells or plant tissues as described in Plant Molecular Biology Manual (Gelvin, S.B. et al., Academic Press Publishers ).
  • plant tissue also includes a callus obtainable by culturing plant cells.
  • a binary vector e.g., pBI121 or pPZP202 may be used.
  • a particle gun a whole plant, a plant organ or a plant tissue may be used directly, or sections may be prepared therefrom before use, or protoplasts may be prepared and used.
  • the thus prepared samples may be treated using a gene transfer device (e.g., PDS-1000 (BIO-RAD)).
  • a gene transfer device e.g., PDS-1000 (BIO-RAD)
  • treatment conditions will vary depending on the type of plant or sample, the treatment is generally conducted at a pressure of about 450 to 2000 psi and at a distance of about 4 to 12 cm.
  • the transformed cells or plant tissues are first selected by drug resistance such as hygromycin resistance, and then regenerated into whole plants in a standard manner. Regeneration from transformed cells into whole plants may be accomplished by techniques known to those skilled in the art as appropriate for the type of plant cells.
  • transformation may be accomplished by introducing a recombinant vector into the cultured cells with a gene gun or by electroporation, etc.
  • Calli, shoots, hairy roots and the like obtained as a result of transformation may be used directly for cell culture, tissue culture or organ culture, and may also be regenerated into whole plants using conventionally known procedures for plant tissue culture, e.g., by being administered with an appropriate concentration of a plant hormone (e.g., auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide).
  • a plant hormone e.g., auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide.
  • Confirmation of whether or not the polynucleotide of the present invention has been introduced into a plant may be accomplished by PCR, Southern hybridization, Northern hybridization, etc.
  • DNA is prepared from a transgenic plant and DNA specific primers are designed for PCR.
  • PCR may be performed under the same conditions as used for preparation of the above plasmid.
  • amplification products may be subjected to, e.g., agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, followed by staining with ethidium bromide, SYBR Green solution, etc. If the amplification products are detected as a single band, it can be confirmed that the plant has been transformed.
  • primers which have been labeled with a fluorescent dye or the like may be used in PCR to thereby detect amplification products.
  • amplification products are bound onto a solid phase (e.g., a microplate) and confirmed by fluorescence or enzymatic reaction, etc.
  • progeny plants may be obtained by sexual or asexual reproduction of the whole plant. Moreover, from such a whole plant or progeny plants thereof or clones thereof, for example, seeds, fruits, cuttings, tubers, root tubers, rootstocks, calli, protoplasts or the like may be obtained and used to achieve mass production of the whole plant.
  • the present invention also encompasses a whole plant into which the polynucleotide of the present invention has been introduced in an expressible form, or progeny plants of the whole plant which have the same properties as the whole plant, or tissues derived from the whole plant and progeny plants thereof.
  • Transgenic plants according to the present invention include plants of the family Solanaceae (e.g., eggplant, tomato, hot pepper, potato, tobacco, stramonium, Chinese lantern plant, petunia, calibrachoa, nierembergia), plants of the family Leguminosae (e.g., soybean, adzuki bean, peanut, kidney bean, broad bean, Bird's foot trefoil), plants of the family Rosaceae (e.g., strawberry, Japanese apricot, cherry tree, rose, blueberry, blackberry, bilberry, cassis, raspberry), plants of the family Caryophyllaceae (e.g., carnation, gypsophila), plants of the family Asteraceae (e.g., chrysanthemum, stevia, gerbera, sunflower, daisy), plants of the family Orchidaceae (e.g., orchid), plants of the family Primulaceae (e
  • Solanaceae e.g.
  • Examples of preferred plants include Camellia sinensis, hop ( Humulus lupulus ), sweet potato ( Ipomoea batatas ), snapdragon ( Antirrhinum majus ), stevia ( Stevia rebaudiana ), Arabidopsis thaliana, grape ( Vitaceae vitis ) and so on.
  • the whole plant transformed with the polynucleotide of the present invention (hereinafter referred to as “the plant of the present invention” or “the whole plant of the present invention”) is rich in hexenol glycosides when compared to the wild-type counterpart.
  • the plant of the present invention or the whole plant of the present invention is rich in glycosides of the substrate of the present invention when compared to the wild-type counterpart.
  • the plant of the present invention can be easily obtained as a perfect whole plant by being grown from a seed, a cuttage, a bulb or the like of the plant of the present invention.
  • the plant of the present invention encompasses a whole plant, a plant organ (e.g., leaf, petal, stem, root, seed, bulb), a plant tissue (e.g., epidermis, phloem, parenchyma, xylem, vascular bundle, palisade tissue, spongy parenchyma) or a cultured plant cell, or alternatively, various forms of plant cells (e.g., suspension cultured cells), a protoplast, a leaf section, a callus and so on.
  • a plant organ e.g., leaf, petal, stem, root, seed, bulb
  • a plant tissue e.g., epidermis, phloem, parenchyma, xylem, vascular bundle, palisade tissue, spongy parenchyma
  • a cultured plant cell e.g., various forms of plant cells (e.g., suspension cultured cells), a protoplast,
  • the present invention also provides an extract of the above transformant. Since the transformant of the present invention is rich in hexenol glycosides when compared to the wild-type counterpart, an extract of the transformant is considered to contain hexenol glycosides at high concentrations.
  • the transformant of the present invention is rich in glycosides of the substrate of the present invention when compared to the wild-type counterpart, and hence an extract of the transformant is considered to contain glycosides of the substrate of the present invention at high concentrations.
  • Such an extract of the transformant of the present invention can be obtained as follows: the transformant is homogenized with, e.g., glass beads, a homogenizer or a sonicator and the resulting homogenate is centrifuged to collect the supernatant.
  • a further extraction step may also be provided in accordance with extraction procedures for hexenol glycosides as mentioned above.
  • the extract of the transformant of the present invention can be provided for use in, e.g., production of aromatics and/or industrial raw materials according to standard practice.
  • the present invention also provides a food, a beverage, an aromatic, a pharmaceutical preparation, an industrial raw material, and/or an aromatic cosmetic product, each containing hexenol glycosides produced by the method of the present invention.
  • a food, an aromatic, a pharmaceutical preparation and/or an industrial raw material, each containing the extract of the transformant of the present invention may be prepared in a routine manner. In this way, such a food, an aromatic, a pharmaceutical preparation and/or an industrial raw material, each containing the extract of the transformant of the present invention, contains hexenol glycosides produced by using the method of the present invention.
  • glycosylation is inhibited when suppressing the expression of a protein endogenously occurring in plants and having glycosylation activity on hexenol.
  • the thus modified plants will contain more volatile hexenol in non-glycosylated form and can be expected to release a stronger green aroma.
  • the present invention therefore provides a plant modified to suppress the expression of a protein having glycosylation activity on hexenol.
  • hexenol glycosyltransferase a protein having glycosylation activity on hexenol (hereinafter referred to as "hexenol glycosyltransferase”) is encoded by a polynucleotide of any one selected from the group consisting of (a) to (e) shown below:
  • polynucleotides (a) to (e) are as defined above in the section "2.
  • Non-human transformant rich in hexenol glycosides are as defined above in the section "2.
  • nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31 is intended in some embodiment to mean "the nucleotide sequence shown in SEQ ID NO: 1 or 3," “the nucleotide sequence shown in SEQ ID NO: 5, 7, 9 or 11,” “the nucleotide sequence shown in SEQ ID NO: 13, 15 or 17,” “the nucleotide sequence shown in SEQ ID NO: 19, 21 or 23,” “the nucleotide sequence shown in SEQ ID NO: 25,” “the nucleotide sequence shown in SEQ ID NO: 27 or 29” or “the nucleotide sequence shown in SEQ ID NO: 31.”
  • amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 is intended in some embodiment to mean "the amino acid sequence shown in SEQ ID NO: 2 or 4," “the amino acid sequence shown in SEQ ID NO: 6, 8, 10 or 12,” “the amino acid sequence shown in SEQ ID NO: 14, 16 or 18,” “the amino acid sequence shown in SEQ ID NO: 20, 22 or 24,” “the amino acid sequence shown in SEQ ID NO: 26,” “the amino acid sequence shown in SEQ ID NO: 28 or 30” or “the amino acid sequence shown in SEQ ID NO: 32.”
  • means to suppress the expression of hexenol glycosyltransferase include substances capable of reducing the expression level of messenger RNA (mRNA) for this enzyme, as exemplified by low molecular compounds, hormones, proteins and nucleic acids.
  • mRNA messenger RNA
  • such a substance may be a nucleic acid capable of suppressing the functions or expression of a gene encoding the above enzyme.
  • nucleic acid examples include hairpin-shaped shRNAs (short hairpin RNAs) or double-stranded RNAs (dsRNAs) which produce siRNAs (small interfering RNAs) for RNA interference (RNAi), as well as antisense nucleic acids, decoy nucleic acids, or aptamers, etc. These inhibitory nucleic acids are able to suppress the expression of the above gene.
  • the target gene to be inhibited which encodes hexenol glycosyltransferase consists of any one of the above polynucleotides (a) to (e), and sequence information can be obtained for each polynucleotide. In the present invention, it is possible to use, as a target region to be inhibited, not only a coding region, but also a non-coding region of the gene encoding hexenol glycosyltransferase.
  • RNA interference is a multi-step process proceeding through a number of stages.
  • dsRNA or shRNA expressed from an RNAi expression vector is recognized by Dicer and cleaved into siRNAs of 21 to 23 nucleotides. These siRNAs are then integrated into an RNAi targeting complex, which is called the RNA-induced silencing complex (RISC), and the complexes between RISC and siRNAs bind to target mRNA containing sequences complementary to the siRNA sequences and thereby cleave the mRNA.
  • RISC RNA-induced silencing complex
  • the target mRNA is cleaved in the center of its region complementary to the siRNA, finally leading to rapid degradation of the target mRNA and reduced protein expression levels.
  • siRNA duplexes are known to be sequences of 21 nucleotides in length, each comprising a 19 bp duplex with an overhang of two uridine residues at the 3'-terminal end ( Elbashir S. M. et al., Genes and Dev, 15, 188-200 (2001 )).
  • a target sequence on mRNA may be selected from the cDNA sequence corresponding to the mRNA sequence.
  • the present invention is not limited to this region.
  • siRNA molecules may be designed on the basis of the criteria well known in the art. For example, as a target segment in target mRNA, it is possible to select a segment covering 15 to 30 contiguous bases, preferably 19 to 25 contiguous bases, preferably starting with AA, TA, GA or CA. siRNA molecules have a GC ratio of 30% to 70%, preferably 35% to 55%. Alternatively, a target sequence for RNAi may be selected as appropriate as described in Ui-Tei K. et al. ((2004) Nucleic Acids Res. 32, 936-948 ).
  • siRNA For introduction of siRNA into cells, it is possible to use, e.g., procedures in which synthesized siRNA is ligated to plasmid DNA and then introduced into cells, or procedures in which double-stranded RNA is annealed.
  • shRNA may also be used for providing RNAi effect.
  • shRNA is an RNA molecule called short hairpin RNA, which has a stem-loop structure because some single-stranded regions form complementary strands with other regions.
  • shRNA may be designed to form a stem-loop structure as a part thereof. For example, assuming that a sequence covering a certain region is designated as sequence A, and a strand complementary to the sequence A is designated as sequence B, shRNA is designed to comprise the sequence A, a spacer and the sequence B linked in this order on a single RNA strand and to have an overall length of 45 to 60 bases. The spacer may also have any length.
  • sequence A is a sequence covering a partial region of the target gene encoding hexenol glycosyltransferase
  • the target region there is no particular limitation on the target region and any region may be selected as a candidate for the target region.
  • the sequence A has a length of 19 to 25 bases, preferably 19 to 21 bases.
  • microRNA may be used to inhibit the expression of hexenol glycosyltransferase.
  • microRNA is an intracellular single-stranded RNA molecule having a length of about 20 to 25 bases and is a kind of ncRNA (non-coding RNA) which is considered to have the function of regulating the expression of other genes. miRNA is generated through processing upon transcription into RNA and is present as a nucleic acid capable of forming a hairpin structure which suppresses the expression of a target sequence.
  • miRNA is also an inhibitory nucleic acid based on RNAi
  • miRNA may also be designed and synthesized in the same manner as in the case of shRNA or siRNA.
  • Expression vectors for RNAi may be readily prepared with a commercially available DNA/RNA synthesizer (e.g., Applied Biosystems model 394) on the basis of pMuniH1 plasmid, pSINsi vector (Takara Bio Inc., Japan), pSIF1-H1 (System Biosciences, Inc.), etc.
  • Examples of expression vectors for RNAi include, but are not limited to, pSPB1876 ( WO2004/071467 ).
  • Expression vectors for RNAi may be prepared by entrusting their preparation to third parties such as Cosmo Bio Co., Ltd. (Japan), Takara Bio Inc. (Japan), Invitrogen, Promega, etc.
  • a method for producing a plant modified to suppress the expression of hexenol glycosyltransferase may comprise the following steps.
  • Step of introducing an expression vector for RNAi e.g., siRNA expression vector or miRNA expression vector
  • an expression vector for RNAi e.g., siRNA expression vector or miRNA expression vector
  • Non-human transformant rich in hexenol glycosides The host plant may be any of a whole plant or a portion thereof, i.e., a plant organ (e.g., leaf, petal, stem, root, seed), a plant tissue (e.g., epidermis, phloem, parenchyma, xylem, vascular bundle, palisade tissue, spongy parenchyma) or a cultured plant cell, or alternatively, various forms of plant cells (e.g., suspension cultured cells), a protoplast, a leaf section, a callus and so on.
  • the type of plant is also as described above in the section "2.
  • Non-human transformant rich in hexenol glycosides The type of plant is also as described above in the section "2.
  • Non-human transformant rich in hexenol glycosides The type of plant is also as described above in the section "2.
  • the host plant used in the above step (1) is a portion of a whole plant, such as a plant organ, a plant tissue, a plant cell, a protoplast, a leaf section or a callus
  • the resulting transformant may be grown in an appropriate environment until a perfect whole plant is formed.
  • techniques for growing a portion of a whole plant into a perfect whole plant reference may be made to the descriptions in the following document: Biochemistry Experiments Vol. 41, An Introduction to Plant Cell Technology, Japan Scientific Societies Press, ISBN 4-7622-1899-5 .
  • the hexenol aglycon Upon cultivation of the thus obtained plant which is modified to suppress the expression of a gene for hexenol glycosyltransferase, the hexenol aglycon can be produced efficiently.
  • RNA interference may be performed on polynucleotides (f) to (j) shown below to thereby prepare a whole plant rich in the substrate of the present invention in non-glycosylated form:
  • the aglycon of the substrate of the present invention can be produced efficiently.
  • a processed product of a plant e.g., natural flower, cut flower
  • a portion of the plant e.g., leaf, petal, stem, root, seed, bulb
  • a processed product include, but are not limited to, pressed flowers, dried flowers, preserved flowers, material flowers, resin-embedded products, etc.
  • Extract of a plant modified to suppress the expression of hexenol glycosyltransferase and use thereof
  • the present invention also provides an extract of the above plant modified to suppress the expression of a gene for hexenol glycosyltransferase or for glycosyltransferase acting on the substrate of the present invention. Since the plant modified to suppress the expression of a gene for hexenol glycosyltransferase or for glycosyltransferase acting on the substrate of the present invention is rich in hexenol aglycon or in the aglycon of the substrate of the present invention when compared to the wild-type counterpart, an extract of the modified plant is considered to contain hexenol aglycon or the aglycon of the substrate of the present invention at high concentration.
  • the above extract can be extracted in the same manner as described above for the extract of the transformant of the present invention.
  • the thus obtained extract can be provided for use in, e.g., production of aromatics and/or industrial raw materials according to standard practice.
  • the present invention also provides an aromatic and/or an industrial raw material, each containing the above extract.
  • an aromatic and/or an industrial raw material, each containing the above extract may be prepared in a routine manner.
  • such an aromatic and/or an industrial raw material, each containing the extract of the plant modified to suppress the expression of a gene for hexenol glycosyltransferase or for glycosyltransferase acting on the substrate of the present invention contains hexenol aglycon or the aglycon of the substrate of the present invention generated by using the plant modified to suppress the expression of hexenol glycosyltransferase or glycosyltransferase acting on the substrate of the present invention.
  • the aromatic and industrial raw material of the present invention are of the same type and composition as described above in the section "3. Extract of transformant and use thereof.” ⁇ Since the relevant detailed descriptions in Section 3. have been deleted, this sentence should also be deleted.
  • the present invention provides a screening method for a plant rich in hexenol aglycon. More specifically, the above method comprises steps (1) to (3) shown below:
  • the above step (1) may be accomplished by extracting mRNA from a test plant. Although mRNA may be extracted from any site of the test plant, preferred are petals. Once mRNA has been extracted, cDNA may be prepared from the mRNA through reverse transcription.
  • the above step (2) may be accomplished as follows: a polynucleotide or oligonucleotide consisting of a nucleotide sequence complementary to the polynucleotide of the present invention is used as a probe or primer and allowed to hybridize with the mRNA extracted above under high stringent conditions. High stringent conditions are as already described above.
  • a polynucleotide or oligonucleotide has a length of preferably 5 to 500 bp, more preferably 10 to 200 bp, and even more preferably 10 to 100 bp.
  • the polynucleotide or oligonucleotide may be readily synthesized with various automatic synthesizers (e.g., AKTA oligopilot plus 10/100 (GE Healthcare)), or alternatively, its synthesis may be entrusted to a third party (e.g., Promega or Takara), etc.
  • various automatic synthesizers e.g., AKTA oligopilot plus 10/100 (GE Healthcare)
  • a third party e.g., Promega or Takara
  • the step (3) may be accomplished by commonly used techniques for detection of hybridization, such as Southern blotting, Northern blotting ( Sambrook, Fritsch and Maniatis, "Molecular Cloning: A Laboratory Manual” 2nd Edition (1989), Cold Spring Harbor Laboratory Press ), microarrays (Affymetrix; see United States Patent Nos.
  • the step (3) may be accomplished by PCR amplification and the subsequent analysis of the resulting amplification products by electrophoresis or sequencing ( Sambrook, Fritsch and Maniatis, "Molecular Cloning: A Laboratory Manual” 2nd Edition (1989), Cold Spring Harbor Laboratory Press ), etc., to detect hybridization.
  • a whole plant in which hybridization was more often detected can be regarded as expressing higher levels of a protein having glycosylation activity on hexenol compounds than other whole plants, and hence such a whole plant is predicted to be rich in hexenol glycosides.
  • a whole plant in which hybridization was less often detected shows lower expression of a protein having glycosylation activity on hexenol compounds than other whole plants, and hence such a whole plant is predicted to be rich in hexenol aglycon and to release a strong green aroma.
  • the present invention provides a composition for producing a hexenol glycoside, which comprises the protein of the present invention and a solvent (hereinafter referred to as "the composition of the present invention").
  • composition of the present invention may further comprise a UDP-sugar.
  • the protein of the present invention a hexenol glycoside and a UDP-sugar are as described above.
  • the solvent used for this purpose is not limited in any way, but preferred is a neutral buffer of pH 6.0 to 8.0 (e.g., sodium phosphate buffer or potassium phosphate buffer).
  • composition of the present invention When the composition of the present invention is mixed and reacted with hexenol (and a UDP-sugar), a hexenol glycoside can be easily produced.
  • reaction of the protein of the present invention with a UDP-sugar and a hexenol molecule is as described above.
  • phages (about 300,000 pfu) from the cDNA library of Camellia sinensis (cultivar Yabukita) (Non-patent Document 4) were used for comprehensive screening of glycosyltransferase genes using Arabidopsis thaliana glycosyltransferase. Fragments amplified with the Arabidopsis thaliana UGT85A3 gene specific primer set (SEQ ID NOs: 33 and 34) and UGT85A1 gene specific primer set (SEQ ID NOs: 35 and 36) were used as screening probes to conduct plaque hybridization screening.
  • the probes were each labeled by PCR using a non-radioisotope DIG-nucleic acid detection system (Roche Diagnostics) under the conditions recommended by the manufacturer.
  • a PCR reaction solution used for this purpose was prepared to contain 1 ⁇ l of template DNA (total cDNA from Arabidopsis thaliana ), 1 ⁇ Taq buffer (TakaRa Bio), 0.2 mM dNTPs, primers (0.2 pmol/ ⁇ l each) and rTaq polymerase (1.25 U).
  • This PCR reaction solution was reacted at 94°C for 5 minutes, followed by 30 cycles of reaction at 94°C for 1 minute, at 52°C for 1 minute and at 72°C for 2 minutes, and final treatment at 72°C for 5 minutes.
  • This PCR product was applied to a Mini Quick Spin column (Roche) to remove the primers and unreacted dNTPs, and the resulting product was used as a screening probe.
  • Cs_UGT Camellia sinensis UGT genes
  • Each Cs_UGT fragment of approximately 1.4 kb was excised by means of the NdeI and XhoI or SalI or BamHI restriction enzyme sites added to the primers, and then ligated to the NdeI and XhoI or BamHI sites of an E. coli expression vector, pET15b (Novagen), to thereby obtain an E. coli expression vector for each enzyme gene.
  • Each vector was designed to carry the open reading frame of the Cs_UGT gene in frame with a His tag located upstream of the NdeI site so as to express a chimeric protein having the His tag fused to the N-terminal end of Cs_UGT.
  • Each cultured transformant was collected by centrifugation (5,000 ⁇ g, 10 min) and then added to and suspended in Buffer S [20 mM HEPES buffer (pH 7.5), 20 mM imidazole, 14 mM ⁇ -mercaptoethanol] at 1 ml/g cell. Subsequently, the suspension was homogenized by ultrasonication (15 sec, repeated 8 times) and then centrifuged (15,000 ⁇ g, 15 min). The resulting supernatant was collected as a crude enzyme solution. The crude enzyme solution was loaded onto a His SpinTrap column (GE Healthcare) which had been equilibrated with Buffer S, followed by centrifugation (70 ⁇ g, 30 sec).
  • Buffer S 20 mM HEPES buffer (pH 7.5), 20 mM imidazole, 14 mM ⁇ -mercaptoethanol
  • Standard enzyme reaction conditions are as follows. A reaction solution (2 mM UDP-glucose, 1.5 mM sugar acceptor substrate, 100 mM potassium phosphate buffer (pH 7.5), 25 ⁇ l purified Cs_UGT enzyme solution) was prepared in a volume of 50 ⁇ l with distilled water and reacted at 30°C for 1 hour.
  • the enzyme reaction solution (5 ⁇ l) was analyzed by LC-MS under the following conditions.
  • glycosyltransferases on green leaf volatiles have a certain correlation between their sequence and functions, and are present in a wide range of land plants.
  • CsUGTC30 isolated from Camellia sinensis was found to have the ability to cause glucosylation of green leaf volatiles (GLV) typified by cis- 3-hexen-1-ol (hexenol).
  • GLV green leaf volatiles
  • Leaves of Camellia sinensis are known to contain not only glycosides of green leaf volatiles, but also monoterpene-based aroma components (e.g., geraniol, linalool) and aromatic-based aroma components in the form of glycosides (Non-patent Document: Wang et al., J. Agric. Food Chem. 2000, 48, 5411-5418 ).
  • CsUGTC30 showed the highest activity on geraniol and showed the second highest activity on eugenol and benzyl alcohol at the same level as on hexenol.
  • CsUGTC30 was also found to have transglycosylation activity on 2-phenylethanol and linalool. In contrast, CsUGTC30 showed no transglycosylation activity on the flavonoids quercetin (flavonol) and cyanidin (anthocyanidin), which are non-aroma components ( Figure 8 : the activity on geraniol is set to 100%).
  • CsUGTC30 has the ability to cause glycosylation not only of green leaf volatiles, but also of monoterpene alcohol-based and aromatic-based aroma components.
  • Non-patent Document 2 Noguchi et al. (2009) Plant Cell, 21: 1556-1572 .
  • geraniol as a sugar acceptor, 4 types of UDP-sugars, i.e., UDP-glucose, UDP-xylose, UDP-galactose and UDP-glucuronic acid were used for enzyme reaction.
  • the reaction time was set to 30 minutes at 30°C.
  • CsUGTC30 showed high specificity for UDP-glucose, and its specificity for UDP-galactose was 15% of that for UDP-glucose ( Figure 9 : the activity on UDP-glucose is set to 100%).
  • CsUGTC30 was found to use UDP-glucose as a major sugar donor.
  • RNAs were extracted from old leaves, mature leaves, young leaves, young leaves (treated in the dark), stems, roots and flowers of Camellia sinensis (cultivar Yabukita), followed by quantitative RT-PCR in the same manner as previously reported (Non-patent Document 3: Ono et al. (2010) Plant Cell, 22: 2856-2871 ). For treatment in the dark, the light was shielded for 24 hours with aluminum.
  • the specific primers for CsUGTC30 (SEQ ID NOs: 37 and 38) and the specific primers for internal standard 18S rRNA (SEQ ID NOs: 39 and 40) used here are as shown below.
  • CsUGTC30 was confirmed to be expressed at the highest level in young leaves ( Figure 10 ). This result is in agreement with the finding that glycosides of aroma components in Camellia sinensis are accumulated in leaves. These results indicate that CsUGTC30 is expressed in young leaves and is an important enzyme responsible for glucosylation of various aroma components to thereby generate watersoluble aroma precursors in leaves.
  • green leaf volatiles are important components determining the quality of the food products. For this reason, there is a demand for techniques to control green leaf volatiles. From a plurality of plant lines, the inventors of the present invention have now found several types of glycosyltransferases having glycosylation activity on green leaf volatiles. When using or controlling these enzymes, a green aroma can be enhanced or reduced.
  • the present invention provides food products with modified aroma or an important tool for development of aromatics.

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MY167872A (en) 2010-06-02 2018-09-26 Evolva Inc Recombinant production of steviol glycosides
WO2013022989A2 (fr) 2011-08-08 2013-02-14 Evolva Sa Production par recombinaison de glycosides de stéviol
CN105051195B (zh) 2013-02-06 2019-09-06 埃沃尔瓦公司 用于提高莱鲍迪苷d和莱鲍迪苷m之产生的方法
AU2014213918B2 (en) 2013-02-11 2017-11-09 Danstar Ferment Ag Efficient production of steviol glycosides in recombinant hosts
SG10201901007PA (en) 2014-08-11 2019-03-28 Evolva Sa Production of steviol glycosides in recombinant hosts
EP3190905A2 (fr) 2014-09-09 2017-07-19 Evolva SA Production de glycosides de stéviol dans des hôtes de recombinaison
EP3250686A1 (fr) 2015-01-30 2017-12-06 Evolva SA Production de glycosides de stéviol dans des hôtes de recombinaison
WO2016146711A1 (fr) 2015-03-16 2016-09-22 Dsm Ip Assets B.V. Udp-glycosyltransférases
MX2018001620A (es) 2015-08-07 2018-06-11 Evolva Sa Produccion de glucosidos de esteviol en hospedadores recombinantes.
EP3387136B1 (fr) * 2015-12-10 2022-01-05 Evolva SA Production de glycosides de stéviol dans des hôtes recombinants
MY198001A (en) 2016-04-13 2023-07-25 Evolva Sa Production of steviol glycoside in recombinant hosts
US10815514B2 (en) 2016-05-16 2020-10-27 Evolva Sa Production of steviol glycosides in recombinant hosts
WO2018083338A1 (fr) 2016-11-07 2018-05-11 Evolva Sa Production de glycosides de stéviol dans des hôtes recombinants
US11447765B2 (en) * 2017-04-12 2022-09-20 Suntory Holdings Limited Rhamnose synthase derived from stevia and gene
CN108070576A (zh) * 2018-02-05 2018-05-25 安徽农业大学 一种茶树糖基转移酶突变体及其在植物害虫防御中的应用
CN109628421B (zh) * 2019-01-11 2022-11-01 安徽农业大学 一种特异合成呋喃酮葡萄糖苷的糖基转移酶及其应用
CA3164769A1 (fr) * 2019-12-16 2021-06-24 Manus Bio, Inc. Production microbienne de mogrol et de mogrosides
CA3177183A1 (fr) * 2020-05-13 2021-11-18 Ginkgo Bioworks, Inc. Biosynthese de mogrosides
CN118222658B (zh) * 2024-05-22 2024-07-26 天津凯莱英生物科技有限公司 生物合成红景天苷的方法

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5925525A (en) 1989-06-07 1999-07-20 Affymetrix, Inc. Method of identifying nucleotide differences
US5858659A (en) 1995-11-29 1999-01-12 Affymetrix, Inc. Polymorphism detection
US6045996A (en) 1993-10-26 2000-04-04 Affymetrix, Inc. Hybridization assays on oligonucleotide arrays
EP1234046B1 (fr) 1999-12-01 2008-11-12 Adelaide Research & Innovation Pty Ltd. Glucose-udp:aglycon-glucosyltransferase
US20040172682A1 (en) 2003-02-12 2004-09-02 Kinney Anthony J. Production of very long chain polyunsaturated fatty acids in oilseed plants
WO2004111254A1 (fr) * 2003-06-19 2004-12-23 Poalis A/S Methode de production d'un compose organique de faible poids moleculaire dans une cellule
JP4481702B2 (ja) 2004-03-31 2010-06-16 サントリーホールディングス株式会社 脂質生産菌の育種方法およびその利用
US9441233B2 (en) 2010-05-06 2016-09-13 Ceres, Inc. Transgenic plants having increased biomass
MY167872A (en) * 2010-06-02 2018-09-26 Evolva Inc Recombinant production of steviol glycosides
EA201400836A8 (ru) * 2012-01-23 2016-03-31 ДСМ АйПи АССЕТС Б.В. Получение дитерпена
JP6153336B2 (ja) * 2012-02-06 2017-06-28 サントリーホールディングス株式会社 チャ由来モノテルペン配糖体化酵素及びその利用方法

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